[CANCER RESEARCH 64, 8987–8993, December 15, 2004] Microarray-based Detection of Multidrug Resistance in Human Tumor Cells by Expression Profiling of ATP-binding Cassette Transporter Genes Jean-Pierre Gillet,1 Thomas Efferth,2 Daniel Steinbach,3 Jacques Hamels,4 Franc¸oise de Longueville,5 Vincent Bertholet,5 and Jose´Remacle1 1Research Unit of Cellular Biology, University of Namur, Namur, Belgium; 2Center for Molecular Biology of the University of Heidelberg, Heidelberg, Germany; 3University Children’s Hospital Jena, Department of Pediatrics, Jena, Germany; 4Genetic and Pathology Institute, Department of Pathology and Molecular Onco-Hematology, Loverval, Belgium; and 5Eppendorf Array Technologies, Namur, Belgium ABSTRACT transporter genes, e.g., ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (MXR/BCRP; refs. 5–7). The parental sensitive cell lines are used as Different mechanisms of drug resistance, including ATP-binding cas- reference and their corresponding drug-resistant subline as test. sette (ABC) transporters, are responsible for treatment failure of tumors. We developed a low-density DNA microarray which contains 38 genes of the ABC transporter gene family. This tool has been validated with three MATERIALS AND METHODS different multidrug-resistant sublines (CEM/ADR5000, HL60/AR, and MCF7/CH1000) known to overexpress either the ABCB1 (MDR1), ABCC1 Cell Lines. Human T-lymphoblastoid leukemic ABCB1 (MDR1) express- (MRP1), or ABCG2 (MXR and BCRP) genes. When compared with their ing CCRF/ADR5000 cells selected with doxorubicin and parental ABCB1 drug-sensitive parental lines, we observed not only the overexpression of (MDR1)-negative CCRF-CEM cells were obtained from Dr. Axel Sauerbrey these genes in the multidrug-resistant cell lines but also of other ABC (Department for Pediatrics, University of Jena, Jena, Germany). These cells transporter genes pointing to their possible role in multidrug resistance. were cultured as described previously (5). These results were corroborated by quantitative real-time reverse tran- Promyelocytic ABCC1 (MRP1)-overexpressing HL60/AR leukemia cells scription-PCR. As the microarray allows the determination of the expres- and parental ABCC1 (MRP1)-negative HL60 drug-sensitive cells were ob- sion profile of many ABC transporters in a single hybridization experi- tained from Dr. Sauerbrey and seeded in RPMI 1640 supplemented with 10% ment, it may be useful as a diagnostic tool to detect drug resistance in fetal calf serum and 100 mmol/L daunorubicin for HL60/AR. Parental HL-60 clinical samples. cells were maintained under the same conditions without daunorubicin expo- sure (6). INTRODUCTION Human breast carcinoma parental MCF7 cell line and the multidrug-resist- ant MCF7/CH1000 subline were kindly provided by Dr. Douglas D. Ross Multidrug resistance (MDR) in tumor treatment is characterized by (University of Maryland Greenebaum Cancer Center, Department of Medicine, resistance to a broad spectrum of structurally unrelated cytotoxic University of Maryland School of Medicine, and the Baltimore Veterans drugs and is of prognostic relevance for treatment outcome (1). Drug Affairs Medical Center; Baltimore, MD; ref. 7). resistance may develop after prolonged exposure to a drug and is The panel of 60 human tumor cell lines of the Developmental Therapeutics mediated by different cellular mechanisms, including the expression Programme of the National Cancer Institute (NCI; Bethesda, MD) consisted of of ATP-binding cassette (ABC) transporters (2). The ABCB1 (MDR1), leukemia, melanoma, non–small cell lung cancer, colon cancer, renal cancer, ABCC1 (MRP1), and ABCG2 (MXR and BCRP) genes are the best ovarian cancer cell lines, cell lines of tumors of the central nervous system, known genes associated with MDR, and at least seven other ABC prostate carcinoma, and breast cancer. Their origin and processing have been described previously (8). transporters are also capable of transporting drugs (2). The presence of ϩ Isolation of mRNA. PolyA RNA was isolated with the FastTrack 2.0 other ABC transporters such as ABCC7 and ABCA4 are linked to other mRNA isolation kit (Invitrogen, Merelbeke, Belgium) with the manufacturer’s diseases such as cystic fibrosis or Stargardt’s disease (3). However, protocol for isolating mRNA starting from 4 ϫ 107 cells. RNA integrity was several ABC transporters have still unknown or undefined functions. verified by capillary electrophoresis on the Agilent 2100 Bioanalyzer (Agilent In recent years, MDR has been diagnosed in tumors by various types Technologies, Palo Alto, CA). of assays. There is actually no technique for obtaining a full picture of Synthesis of Labeled cDNA. Labeled cDNA were prepared with 1 ␮gof the different MDR parameters but mostly determination of individual mRNA. Three synthetic polyAϩ-tailed RNA samples were spiked at three genes or proteins of the ABC family. High-density DNA microarray different amounts (10, 1, and 0.1 ng per reaction) into the purified mRNA as analysis have been done to analyze differentially expressed genes in internal standard to assist in quantification and estimation of experimental tumor cells after drug treatment (4). Although the use of high-density variation introduced during labeling and analysis. The detailed procedure was already reported previously (9). DNA microarray is very useful for the screening of new markers, the Design of Low-Density Microarrays for 38 ABC Transporter Genes. quantitation of gene expression is rather poor. Low-density DNA The genes on the low-density microarray (termed DualChip human ABC) are microarrays are more suitable for routine applications because of their presented in Table 1. It contained two arrays per slide with a range of 41 simplicity, good reproducibility, easy data management, and low transporter genes composed of 38 ABC transporters, 1 cationic transporter, and costs. The aim of the present study was to test a novel low-density 2 ATP-sensitive potassium channels. To evaluate the reliability of the exper- microarray for the simultaneous expression analysis of 38 ABC trans- imental data, several positive and negative hybridization and detection controls porter genes in multidrug-resistant tumor cells. To investigate the are included on the microarray. Three internal standard controls and eight usefulness of such a new tool, we investigated three multidrug- housekeeping genes are arrayed on the slides for the normalization. DualChip resistant sublines that are known to express three different ABC human ABC is composed of single-strand DNA probes attached to the glass support by a covalent link. Each DNA probe is present in triplicates (Fig. 1A). The length of the DNA probes has been optimized, and the design of the probes Received 6/4/04; revised 9/16/04; accepted 10/13/04. has been done as described previously. Recent update and the absence of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with several clones, 11 ABC transporters are absent on the array. The homology 18 U.S.C. Section 1734 solely to indicate this fact. between the different genes of this superfamily is very high. It is 35 to 40% in Note: J-P. Gillet and T. Efferth contributed equally to this work. average and for certain genes Ͼ60 to 70%. For this reason, in five cases, the Requests for reprints: Jean-Pierre Gillet, Research Unit of Cellular Biology, University capture-probe was complementary of two closely related genes (ABCA2/3, of Namur, Rue de Bruxelles 61, 5000 Namur, Belgium. Phone: 32-081-72-57-11; Fax: 32-081-72-41-35; E-mail: [email protected]. ABCB1/4, ABCC6/8/9, and Kir 6.1/6.2). The specificity of the capture-probe ©2004 American Association for Cancer Research. was checked by testing the binding of PCR-amplified ABC transporter clones. 8987 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2004 American Association for Cancer Research. DETECTION OF ABC TRANSPORTERS BY MICROARRAYS Table 1 Human ABC genes: their representation on the DualChip human ABC, chromosomal loci, expression and function Family Members On ABCChips Location Expression Function ABC A ABC A1 Yes 9q31.1 Ubiquitous Cholesterol efflux onto HDL ABC A2 Yes 9q34.3 Brain Drug resistance ABC A3 Yes 16p13.3 Lung Surfactant secretion ABC A4 Yes 1p21.3 Rod photoreceptors N-Retinylidiene-PE efflux ABC A5 Yes 17q24.3 Muscle, heart, testes ABC A6 Yes 17q24.3 Liver ABC A7 Yes 19p13.3 Spleen, thymus ABC A8 Yes 17q24.3 Ovary ABC A9 No 17q24.3 Heart ABC A10 No 17q24.3 Muscle, heart ABC A12 No 2q34 Stomach ABC A13 No 7p12.3 Low in all tissues ABC B ABC B1 Yes 7q21.12 Adrenal, kidney, brain Multidrug resistance ABC B2 Yes 6p21.3 All cells Peptide transport ABC B3 Yes 6p21.3 All cells Peptide transport ABC B4 Yes 7q21.12 Liver Phosphatidylcholine transport ABC B5 No 7p21.1 Ubiquitous ABC B6 Yes 2q35 Mitochondria Iron transport ABC B7 Yes Xq21–q22 Mitochondria Fe/S cluster transport ABC B8 Yes 7q36.1 Mitochondria ABC B9 Yes 12q24.31 Heart, brain ABC B10 Yes 1q42.13 Mitochondria ABC B11 Yes 2q24.3 Liver Bile salt transport ABC C ABC C1 Yes 16p13.12 Lung, testes Multidrug resistance ABC C2 Yes 10q24.2 Liver Organic anion efflux ABC C3 Yes 17q21.33 Lung, intestine, liver Drug resistance ABC C4 Yes 13q32.1 Prostate Nucleoside transport ABC C5 Yes 3q27.1 Ubiquitous Nucleoside transport ABC C6 Yes 16p13.12 Kidney, liver ABC C7 Yes 7q31.31 Exocrine tissues Chloride ion channel ABC C8 Yes 11p15.1 Pancreas Sulfonylurea receptor ABC C9A